Project Summary Intracellular bacterial pathogens cause a large number of diseases of public health importance. Their intracellular cycle is key to virulence and culminates in the biogenesis of a niche dedicated to their survival, proliferation or persistence, a feat achieved via their subversion of host cell functions. Determining the mechanisms used to create these niches is critical to understanding their pathogenesis, providing broad insights into pathogenic themes shared by microbes. Bacterial subversion of host cell functions invokes delivery of effector proteins, whose modes of action hold keys to their pathogenic mechanisms. Bacteria of the genus Brucella, the causative agents of the world-widespread zoonosis brucellosis, generate a replication-permissive organelle, the Brucella-containing vacuole (rBCV), which is essential to their pathogenesis and derived from the host endoplasmic reticulum (ER). rBCV biogenesis requires the Brucella VirB Type IV secretion system (T4SS) and host secretory trafficking, suggesting it is controlled by T4SS-delivered effector proteins that modulate specific secretory functions. Yet, the bacterial effectors and host factors of rBCV biogenesis are mostly unknown. We recently discovered Brucella VirB T4SS effectors (BspA, BspB and BspF) that target the host secretory pathway, contribute to either rBCV biogenesis or bacterial growth within rBCVs, and interact with various host proteins functionally associated with either ER-associated degradation (ERAD) or secretory trafficking at the Golgi apparatus. These findings provide timely opportunities to address outstanding questions about the molecular mechanisms of rBCV biogenesis. Here we propose to elucidate the mechanisms by which the newly identified T4SS effector proteins BspA, BspB and BspF modulate distinct host secretory functions to promote rBCV biogenesis and bacterial proliferation. We will use molecular and cellular approaches to first dissect the mode of action of BspB in modulating Golgi-associated secretory trafficking via its interaction with the COG complex, and define how it contributes to rBCV biogenesis. Second, we will determine whether BspF targeting of optineurin (OPTN)-mediated functions in post-Golgi vesicular trafficking mediates its role in Brucella intracellular replication. Last, we will examine whether BspA modulates ER-associated degradation (ERAD) via its targeting of the ERAD E3 ubiquitin ligase MARCH6 to facilitate rBCV biogenesis and Brucella proliferation. The successful completion of this project will provide the first characterizations of Brucella effectors involved in rBCV biogenesis via targeting of host secretory functions. By identifying novel host functions involved in the Brucella intracellular cycle, the proposed research will define new paradigms of pathogenic exploitation of the host secretory pathway applicable to the many intracellular microbes that target this cellular compartment, therefore having a broad impact on the research areas of microbial pathogenesis, cell biology and innate immunity.